BackgroundThe study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents.ResultsIn this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination.ConclusionsThese results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0087-z) contains supplementary material, which is available to authorized users.

OBJECTIVES:Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn's disease (CD).METHODS:Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics.RESULTS:Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN.CONCLUSIONS:Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.

BackgroundFoodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella.ResultsWe demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h.ConclusionsBoth approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0677-2) contains supplementary material, which is available to authorized users.

Limiting microbial growth during drinking water distribution is achieved either by maintaining a disinfectant residual or through nutrient limitation without using a disinfectant. The impact of these contrasting approaches on the drinking water microbiome is not systematically understood. We use genome-resolved metagenomics to compare the structure, metabolic traits, and population genomes of drinking water microbiome samples from bulk drinking water across multiple full-scale disinfected and non-disinfected drinking water systems. Microbial communities cluster at the structural- and functional potential-level based on the presence/absence of a disinfectant residual. Disinfectant residual alone explained 17 and 6.5% of the variance in structure and functional potential of the drinking water microbiome, respectively, despite including multiple drinking water systems with variable source waters and source water communities and treatment strategies. The drinking water microbiome is structurally and functionally less diverse and variable across disinfected compared to non-disinfected systems. While bacteria were the most abundant domain, archaea and eukaryota were more abundant in non-disinfected and disinfected systems, respectively. Community-level differences in functional potential were driven by enrichment of genes associated with carbon and nitrogen fixation in non-disinfected systems and γ-aminobutyrate metabolism in disinfected systems likely associated with the recycling of amino acids. Genome-level analyses for a subset of phylogenetically-related microorganisms suggests that disinfection selects for microorganisms capable of using fatty acids, presumably from microbial decay products, via the glyoxylate cycle. Overall, we find that disinfection exhibits systematic selective pressures on the drinking water microbiome and may select for microorganisms able to utilize microbial decay products originating from disinfection-inactivated microorganisms.